Like eukarya, bacteria and archaea have evolved a variety of defense systems to protect themselves from selfish genetic elements (phages, transposons, and plasmids). On the other hand, successfully disseminated mobile elements can benefit the bacterial hosts, for instance, by introducing antibiotics resistant genes. Understanding the defense systems, therefore, not only offers insights on prokaryotic molecular biology principles but may also provide new strategies to defend the antibiotics resistance epidemics. Recently, a novel widespread defense system that functions on a completely different principle than those previously known was discovered. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are found in 40% of bacterial and 90% of archaeal genomes that confers a small RNA-based prokaryotic immunity system. In this remarkable process, DNA short sequences capturing the memories of past infection are transcribed and processed into small RNA molecules that then pattern with proteins to silence the new invader nucleic acids. This proposal will investigate the molecular mechanisms of two currently identified CRISPR machineries, that for production of the CRISPR RNAs and that for silencing invading RNA. Experimental aims are designed to provide a comprehensive understanding of the structural and functional properties of the processing endonuclease and the RNA silencing complex. Relevance: The CRISPR elements of bacteria and archaea provide an important vehicle for maintaining a balance in microbial environments through exchange and destruction of genetic materials. CRISPR elements are found in medically important bacteria that include but not limited to Yersinia pestis, Mycobacterium tuberculosis, Haemophilus influenzae, Helicobacter pylori, Neisseria meningitides, Vibrio vulnificus, Staphylococcus aureus, Salmonella Typhi and Clostridium tetani. A thorough understanding of the CRISPR immunity has important implications in studies of bacterial pathogen spread, antimicrobial resistance, and host-virus interactions. The proposed molecular mechanisms of this novel pathway may be exploited for building specific immunity against undesirable genetic elements such as those spreading antibiotics resistance genes. PUBLIC HEALTH RELEVANCE: Prokaryotes and archaea, including many human pathogens, employ a small RNA-based mechanism to maintain environmental balance through exchange and destruction of genetic materials. Structural studies are proposed to study the biochemical mechanism of this pathway.